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Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles.

ABSTRACT: Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.

SUBMITTER: Binder HM

PROVIDER: S-EPMC8699161 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PurposeThe bone marrow niche plays an important role in leukemia development. However, the contributions of different niche components to leukemia development and their underlying mechanisms remain largely unclear.MethodCre/LoxP-based conditional knockout technology was used to delete VPS33B or ANGPTL2 gene in niche cells. Murine B-ALL model was established by overexpressing the N-Myc oncogene in hematopoietic stem progenitor cells. The frequency of leukemia cells and immunophenotypic B220+ CD43+ LICs was detected by flow cytometry. SEVs was isolated by sequential centrifugation and mass spectrometry was performed to analyze the different components of SEVs. Immunoprecipitation and western blot were used to measure the interaction of VPS33B and ANGPTL2.ResultsHere, we showed that specific knockout of vascular protein sorting 33b (Vps33b) in endothelial cells (ECs), but not megakaryocytes or mesenchymal stem cells, resulted in a significant decrease in the secretion of small extracellular vesicles (SEVs) and a delay in the development of B-cell lymphoblastic leukemia (B-ALL). Vps33b knockdown endothelial cells contained much lower levels of SEVs that contained angiopoietin-like protein 2 (ANGPTL2) than the control cells. Importantly, conditional knockout of Angptl2 in ECs significantly delayed B-ALL progression. Moreover, C-terminal region of ANGPTL2 (aa247-471) could directly interact with Sec1-like domain 1 of VPS33B (aa1-aa146). We further demonstrated that the point mutations R399H and G402S in ANGPTL2 led to a dramatic decrease in the secretion of ANGPTL2-SEVs. We also showed that wild-type ANGPTL2-containing SEVs, but not mutant ANGPTL2-containing SEVs, significantly enhanced B-ALL development.ConclusionIn summary, our findings indicate that the secretion of ANGPTL2-containing SEVs in ECs sustains the leukemogenic activities of B-ALL cells, which is fine-tuned by the direct interaction of VPS33B and ANGPTL2. These findings reveal that niche-specific SEVs play an important role in B-ALL development.

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Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles.

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